System and method for performing polynucleotide separations using liquid chromatography

ABSTRACT

Improved liquid chromatography systems having components made of titanium, stainless steel, or organic polymeric material are useful in the separation of polynucleotide fragments, particularly large fragments of double-stranded polynucleotides, by Matched Ion Polynucleotide Chromatography (MIPC). The titanium, stainless steel, or polymeric components are treated so that they do not release multivalent cations into aqueous solutions flowing through the chromatography system. Alternatively, or in addition to utilizing materials made of the components listed above, a multivalent cation capture resin placed upstream of the separation column can be employed to remove multivalent ions from the system. The multivalent cation capture resin can be contained in a guard disk, a guard column, or a guard cartridge. Novel methods for separating mixtures of polynucleotide fragments into fractions based on their molecular weight by Matched Ion Polynucleotide Chromatography and slalom chromatography utilize the liquid chromatographic systems described above.

RELATIONSHIP TO COPENDING APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 09/828,346filed Apr. 5, 2001, now U.S. Pat. No. 6,491,821 which is a continuationof U.S. patent application Ser. No. 09/350,774 filed Jul. 9, 1999 (nowU.S. Pat. No. 6,251,272), which is a continuation of U.S. patentapplication Ser. No. 09/081,040 filed May 18, 1998 (now U.S. Pat. No.5,997,742), which is a continuation-in-part of U.S. patent applicationSer. No. 08/748,376 filed Nov. 13, 1996 (now U.S. Pat. No. 5,772,889).

FIELD OF THE INVENTION

The present invention is directed to the separation of polynucleotidefragments by liquid chromatography. More specifically, the invention isdirected to a liquid chromatography system and method, such as MatchedIon Polynucleotide Chromatography or slalom chromatography, whichenhances the separation of polynucleotides.

BACKGROUND OF THE INVENTION

Separation of polynucleotides is a focus of scientific interest, andnumerous researchers have been attempting to achieve technicalimprovements in various aspects of polynucleotide separation. Anionexchange separation and reverse phase ion pair chromatography are amongthe most frequently used methods for separating polynucleotides.

Previous work has focused on developing rapid, high resolutionseparations, developing separations based on the size of thepolynucleotide fragment rather than the base sequence of the fragment,and on developing the ability to collect fractions of polynucleotides.

W. Bloch (European patent publication No. EP 0 507 591 A2) demonstratedthat, to a certain extent, length-relevant separation of polynucleotidefragments was possible on nonporous anion exchangers withtetramethylammonium chloride (TMAC) containing mobile phases. Y. Ohimyaet al. (Anal. Biochem., 189:126-130 (1990)) disclosed a method forseparating polynucleotide fragments on anion exchange material carryingtrimethylammonium groups. Anion exchangers with diethylaminoethyl groupswere used by Y. Kato et al. to separate polynucleotide fragments (J.Chromatogr., 478:264 (1989)).

An important disadvantage of anion exchange separations ofdouble-stranded polynucleotides is the differing retention behavior ofGC- and AT-base pairs. This effect makes separation according tomolecular size impossible. Another important drawback of the anionexchange methodology is the necessity to use salts and buffers forelution, thus making subsequent investigation of the polynucleotidemolecule fractions very difficult.

U.S. Pat. No. 5,585,236 (1996) to Bonn et al. describes a method forseparating polynucleotides using what was characterized as reverse phaseion pair chromatography (RPIPC) utilizing columns filled with nonporouspolymeric beads. High resolution, rapid separations were achieved usingan ion-pairing reagent, triethylammonium acetate, and acetonitrile/waterreagent mobile phase gradient. This work is important because it is thefirst separation to give size-dependent, sequence-independent separationof double-stranded polynucleotides by chromatography. These separationsare comparable to gel electrophoresis-compatible separations, currentlythe most widely used technology for polynucleotide separations. Bonn'swork makes it possible to automate separations based on the size or onthe polarity of polynucleotides.

In the course of our work on separation of polynucleotides using themethod developed by Bonn et al., with HPLC instrumentation and columnsas described by Bonn, we discovered a degradation effect on theseparation of double-stranded polynucleotides after long-term columnusage (i.e., greater than about 50 injections). This degradation effecthas been generally observed as a loss of resolution for base pairsgreater than 200, as illustrated in the chromatogram of FIG. 1. As thedegradation worsens, increasingly short fragments of polynucleotides areaffected, as shown in FIG. 2. Eventually, the polynucleotides do notelute from the system. As such, the degradation effect or decreasingresolution appears to be a function of the length of the polynucleotidefragment being separated.

There is no published chemical mechanism which would explain such adegradation effect that distinguishes between different size fragmentswhile using reverse phase chromatography. Therefore, we first examinedour procedure for packing the column. We realized that the moleculesthat we were attempting to separate were several magnitudes larger insize than those conventionally separated by reverse phase ion pairliquid chromatography. We suspected that hydrodynamic flow through thecolumn was adequate for short polynucleotide fragments, but was beingdisrupted for larger fragments. In other words, perhaps the longestfragments were being partially sheared. However, we were unable toidentify a packing procedure that would discriminate between short andlong fragments of polynucleotides.

Although we could not conceive a mechanism by which chemicalcontamination could produce these unusual results, we neverthelessexamined contamination of one or more of the various “pure” reagentsemployed in liquid chromatography. After testing each of the reagentsfor contamination, we determined that this was not the source of theproblem. This is not surprising, since the mobile phases used are notcorrosive.

Subsequent clean-up of the column with injections of tetrasodiumethylenediaminetetraacetic acid (EDTA), a metal-chelating agent, largelyrestored chromatographic resolution, as shown in FIG. 3. Putting achelating additive into the mobile phase can provide some protection tothe column. Without wishing to be bound by theory, there are severalmechanisms by which a chelating reagent can provide protection orrestore the instrument or column. One mechanism is the chelating reagentbinds the free metal ions in solution, thus preventing any interactionof the metal ions with the DNA. Another mechanism is the chelatingreagent coats colloidal metal ions, thereby preventing interaction ofthe colloidal metal ions with the DNA. The colloidal metal can beintroduced from the mobile phase, injected into the mobile phase, or canbe released from wetted surfaces in the fluid path. If the chelatingreagent is water soluble, it can eventually dissolve the colloidalmetals.

We were successful in adding small amounts (i.e., 0.1 mM) of tetrasodiumEDTA to the mobile phase without significant changes to thechromatography. However, this concentration of EDTA was not sufficientto protect the columns in all of the stainless steel HPLC instrumentsand columns that were tested. There can be cases where the amount ofmetal ions present or generated are at a concentration where adding achelating reagent will coat or bind the metal ions. In these cases,addition of a small amount of chelating reagent can allow the successfulseparation of DNA fragments.

We tested the use of larger amounts of chelator additive in the mobilephase and found that addition of 10 mM of tetrasodium EDTA impaired theseparation of polynucleotides. It was still uncertain that this higherconcentration of chelating agent provided an acceptable protectivebenefit. While use of EDTA injected into the mobile phase (via the HPLCsample injection valve) demonstrated that the column can be regenerated,addition of chelating agents to the mobile phase is not an idealsolution to the problem as it can hamper subsequent use or analysis ofthe polynucleotide fragments.

We then discovered that placing a cation exchange resin in the flow pathof the mobile phase removed the problem. Guard disks were preparedcontaining a gel-type iminodiacetate resin with an ion exchange capacityof 2.5 mequiv/g (tested with Cu(II)). FIG. 4 shows a chromatogramobtained when the guard disk was positioned directly in front of thesample injection valve. FIG. 5 shows a chromatogram obtained when theguard disk was placed directly in front of the separation column (i.e.,between the injection valve and the column). Attempts to separatepolynucleotides on the stainless steel HPLC system without the use ofguard disks or guard columns containing cation exchange resin orchelating resin resulted in rapid deterioration of the chromatographicseparation.

From the improved results obtained by placing a cation exchange resin inthe flow path of the mobile phase, we concluded that whatever wascausing the peak distortion, probably ionic contaminants, was capable ofbinding to the cation exchange resin. Whatever was causing the fragmentsize-dependent distortion of the peaks had been removed by the cationexchange resin.

Ionic contamination of the system can logically originate in one or moreof several sources. The most significant sources of metal ions are HPLCcomponents containing fritted filters made of stainless steel. Frittedfilter components are used in mobile phase filters, check valve filters,helium spargers, mobile phase mixers, in-line filters, column frits, andother parts of the HPLC. Frits are commonly located at each end of aseparation column in order to contain the particulate packing materialwithin the column. The frit at the head of a column also serves to trapparticulate material. Trapped particulate materials can be metal ionsreleased from another part of the liquid chromatography system. Thelarge surface area associated with any particular fritted component cancontribute to faster solubilization of metals and release of ions. Thus,the ionic contamination from a fritted component can arise in at leasttwo ways. First, the component can be a source of ionic material.Second, it can be a means for trapping ionic material.

Ionic contamination from metals can exist in two forms. One form isdissolved metal ions. In another form, metals ions can exist in thecolloidal state. For example, colloidal iron can be present, even in“high purity” 18 megohm water. Any metal or other ion that can interactwith polynucleotides in the manner described could cause potentiallyharmful chromatographic effects when the metal becomes trapped on thechromatographic column. Magnesium and/or calcium and other ions can bepresent in samples such as PCR products. However, at the concentrationstypically used, magnesium ions present in PCR products do not harm thepeak separation.

Metal ion contamination such as colloidal iron can be released fromfrits, travel to other parts of the HPLC and then be trapped. Thesetypes of contaminants will interfere with DNA in solution or afterhaving been released and trapped on a critical component of the HPLCsuch as the column, an inline filter in front of the detector, or at aback pressure device located after the detector.

In order to test our hypothesis that soluble metals and, potentially,other ions were causing loss of peak resolution during polynucleotideseparations, we challenged the HPLC system with iron, chromium, andnickel. Known concentrations of these three metal ions were added to apolynucleotide standard (pUC18 DNA-Hae III digest). Thepolynucleotide/metal ion solutions were then injected into the HPLC.

Chromium (III) ions (prepared from CrK(SO₄)₂ did not degrade theseparation when present in the sample at 9 mM. However, the samplecontained 100 mM EDTA as a preservative against enzymatic degradationduring storage, and much of the chromium could have been bound in anEDTA complex. However, when chromium was present at 90 mM, fragmentsize-dependent degradation of peaks occurred. At 900 mM chromium, nopeaks could be detected. Several hours later, a sample containing 50 mMCr(III) showed complete loss of the separation peaks.

The same protocol using Ni(II) (prepared from Ni₂SO₄) showedsubstantially no effect on peak shape, although some peak broadening wasobserved at 0.1 M Ni(II).

With Fe(III) (prepared from FeNH₄(SO₄)₂), the effect was less than withCr(II). An injection of 900 μM of Fe(III) in the polynucleotide standardshowed no effect. However, an injection of 2700 μM resulted in a loss ofall peaks. There was some indication that the results weretime-dependent, with the full effect becoming apparent several minutesafter preparation of the metal/polynucleotide sample.

The contact times and metal concentrations of the experiments describedabove were several orders of magnitude higher than would be found in astainless steel HPLC system used for polynucleotide separations. Also,none of the experiments indicated how any reaction could be dependent onthe size of the polynucleotide fragment. However, these data show therelative effect on separations of some of the metals found in stainlesssteel on polynucleotide separation.

As an example of the effects of stainless steel, placement of apreviously used stainless steel frit as an inline frit in front of thecolumn resulted in no peaks being eluted from the column, even aftershort exposure of the frit to the fluid path. In this case all of theDNA was lost in the separation. This means that either DNA was taken upby the frit, or the frit released material that either disrupted theseparation of DNA on the column or within the fluid path to the columnand detector.

The effect of metals on the reverse phase column separation ofpolynucleotides or an effect that discriminated according to fragmentsize has not been reported in the literature. There are, in fact, only alimited number of publications on the chromatographic separation ofpolynucleotides; most of which focus on single-stranded polynucleotides.Separation of single-stranded polynucleotides has been performedroutinely by many workers, but this is usually on very short lengths ofpolynucleotide fragments (usually less than 100-mer, with 25-mer theaverage length), where, based on our observations of double-strandedpolynucleotides, we would expect the degradation effect to be much lesspronounced.

Gunther Bonn and his colleagues have developed the world's leadingchromatographic method for separating double-stranded polynucleotides.Bonn's work was performed on a stainless steel HPLC system withstainless steel hardware, including stainless steel frits. Based on ourdiscovery, we concluded that the effect of metal contamination onpolynucleotide separations was never reported by Bonn or others becausethe amount of dissolved and particulate metals in their stainless steelsystems was below the threshold where degradation of the separationoccurs and the systems worked adequately to produce good peakseparations. Also, our work was carried out over a longer period,perhaps giving sufficient time for accumulation of contaminants withinthe system.

Metal-free or titanium instrumentation is commonly used in proteinseparations, for reasons peculiar to the art of protein separation. Forexample, the activity of a protein can be affected if a metal ispresent. If the protein is intended to be collected and studied,separation is generally performed in a metal-free environment. Also,protein separations use particular mobile phases that can be corrosiveto stainless steel HPLC equipment.

Although metal-free or titanium systems are generally used in theseparation of proteins for the reasons discussed above, it has beendemonstrated that the use of metal-free or titanium systems is notnecessary to maintain the integrity of the separation and that stainlesssteel HPLC systems show equivalent performance (Herold, M. et al.,BioChromatography, 10:656-662 (1991)). In fact, Hewlett-Packard, one ofthe leading manufacturers of HPLC systems, now recommends stainlesssteel systems for use in protein separations.

Because of the success of using stainless steel components in proteinseparations, and because the use of stainless steel systems forpolynucleotide separations had been shown to be successful in the past,there had previously been no indication of the requirement to usenon-metal or titanium system components for liquid chromatographicseparation of polynucleotide fragments.

Our subsequent experiments showed that even if titanium or PEEK fluidpath components are used, then some treatment was necessary before thecomponents could be used for Matched Ion Polynucleotide Chromatography.Although an improvement, our initial use of titanium frits did not giveconsistent results. Treatment of the frits with dilute nitric acid andthen with a chelating agent did improve the performance of theinstrument. Similarly, as shown in the examples, PEEK frits were notconsistently suitable for MIPC, but acid treatment did improve theirperformance.

Finally, degassing the fluid before it enters the liquid chromatographysystem removes the oxygen. This process will inhibit the oxidation andproduction of metal ions in stainless steel or titanium or other tubingcontaining iron. The use of a degasser to remove oxygen can help theMIPC separation. This is probably because the need for an ioncontaminant free fluid path is much more critical in MIPC than in priorart separation processes. The use of the precautions of the method andsystem of the present invention has been found to be much more criticalfor double stranded DNA than for single stranded DNA separations.

As Bonn and coworkers demonstrated, stainless steel can be an excellentmaterial to be used for the fluid path of DNA separations. However, itis difficult to keep the stainless steel surface free of contaminantswhich interfere with MIPC, especially as the surfaces age.

SUMMARY OF THE INVENTION

It is an object of the present invention to achieve optimum peakseparations during the separation of polynucleotides (e.g.double-stranded polynucleotides such as dsDNA) using Matched IonPolynucleotide Chromatography or slalom chromatography.

It is another object of the invention to extend the maximum useful lifeof a chromatographic separation column by protecting the column from thepotentially deleterious effects of ionic contaminants present within theliquid chromatography system.

The invention is a system and method for separating polynucleotidefragments whereby the effects of metal or other ionic contamination areavoided. Although the exact mechanism of the degradation effect remainsunknown, we have determined that, by avoiding stainless steel or othermetal components that can react with phosphate and/or nitrogen groups orunknown groups of the polynucleotides, we are able to separatepolynucleotide fragments to high resolution. This is accomplished by anycombination of several measures: use of non-metal or titanium frits inthe column; use of non-metal or titanium frits in the HPLC system; useof an ion-binding material upstream of the sample injection valve and/orseparation column; use of non-metal or titanium system components (e.g.,pump heads, tubing, pulse dampeners) anywhere within the system thatmobile phase comes in contact with a surface; use of a pre-treatment,non-limiting examples of which include an acid wash treatment, a coatingtreatment, or treatment with a chelating reagent of any componentsurface anywhere within the system that comes into contact with mobilephase in order to eliminate multivalent cation contamination arisingfrom the surface; use of a mobile phase additive that can capture orbind metals or other ions; use of a degassing system for removing oxygenfrom the mobile phase for preventing oxidation of metals in the fluidpath.

In one aspect, the present invention discloses an improved liquidchromatography system for separating a mixture of polynucleotidefragments. The separation can be based on the size or the polarity ofthe fragments. The fragments can be double stranded. The fragmentscomprise at least 5 base pairs. The polynucleotide fragments can becovalently coupled to a detectable tag such as radioisotopes andfluorescent dyes. In a preferred embodiment, the system comprises achromatographic column containing a separation bed of Matched IonPolynucleotide Chromatography (MIPC) separation particles held in thecolumn between porous frits positioned at each end of the column. Thecolumn has an inlet, an injection valve which is in communication withthe inlet by means of a flow path, and mobile phase supply means whichis (are) in communication with the injection valve by means of at leastone flow path. Multivalent cation capture resin capable of removingmultivalent cations from aqueous solutions is positioned in the flowpath. Using the system of the invention, any multivalent cationcontaminants in the flow path are removed before they contact theseparation bed.

The multivalent cation capture resin can be cation exchange resin orchelating resin, but is preferably cation exchange resin having an ionexchange moiety selected from the group consisting of iminodiacetate,nitriloacetate, acetylacetone, arsenazo, hydroxypyridinone, and8-hydroxyquinoline groups. Cation exchange resin having animinodiacetate group is particularly preferred. The multivalent cationcapture resin is preferably contained in a guard disk, guard column, orguard, and is preferably positioned in the flow path between the mobilephase supply means and the injection valve. The system further can alsoinclude multivalent cation capture resin (preferably contained in aguard disk) positioned in the flow path between the injection valve andthe separation column.

The components of the system have process solution-contacting surfaceswhich contact process solutions held within the components or flowingthrough the components. The process solution-contacting surfaces arepreferably material which does not contain or release multivalentcations. The material is most preferably titanium, coated stainlesssteel, or organic polymer. The surfaces are preferably subjected to amultivalent cation removal treatment so that they do not releasemultivalent cations. The treatment can include contacting the surfaceswith aqueous solution containing nitric acid, phosphoric acid,pyrophosphoric acid, or chelating agents. In one embodiment, the processsolutions include a mobile phase additive (e.g. EDTA) that can captureor bind multivalent cation contaminants. The process solutionspreferably are exposed to a degassing system for removing oxygen.

In another aspect, the system comprises a chromatographic columncontaining a separation bed of MIPC separation particles held in thecolumn between porous frits positioned at each end of the column. Thecolumn has an inlet, an injection valve which is in communication withthe inlet by means of a conduit, and mobile phase supply means which is(are) in communication with the injection valve by means of at least oneconduit. The frits have process solution-contacting surfaces which aremade of material which does not release multivalent cations into aqueoussolutions flowing through the frits or collect material from othersources. The material is most preferably titanium, coated stainlesssteel, or organic polymer. The surfaces preferably are subjected to amultivalent cation removal treatment. The treatment can includetreatment with nitric acid, phosphoric acid, pyrophosphoric acid, orchelating agents. In one embodiment, the process solutions include amobile phase additive (e.g. EDTA) that can capture or bind multivalentcation contaminants. The process solutions preferably are exposed to adegassing system for removing oxygen.

The process solution-contacting surfaces of other system components(such as the chromatographic column, injection valve, mobile phasesupply means, and conduits) are also preferably material which does notcontain or release multivalent cations. The system also preferablyincludes multivalent cation capture resin positioned between the mobilephase supply means and the injection valve. The multivalent captureresin is preferably cation exchange resin or chelating resin, which ispreferably contained in a guard column or guard cartridge. The systemcan also include multivalent cation capture resin, preferably containedin a guard disk, positioned between the injection valve and theseparation column.

Also disclosed herein are methods for improving the separation ofpolynucleotide fragments into fractions during MIPC using a liquidchromatographic column containing a separation bed comprising MIPCseparation particles. The separation can be based on the size orpolarity of the fragments. One method comprises supplying and feedingsolutions entering the liquid chromatographic separation column withcomponents having process solution-contacting surfaces which are made ofmaterial which does not release multivalent cations into aqueoussolutions held therein or flowing through the column. The processsolution-contacting surfaces of the components are preferably titanium,coated stainless steel, or organic polymer. The surfaces are preferablysubjected to a multivalent cation removal treatment. The treatment caninclude contacting the surface with an aqueous solution containingnitric acid, phosphoric acid, pyrophosphoric acid, or chelating agents.In one embodiment, the process solutions include a mobile phase additive(e.g. EDTA) that can capture or bind multivalent cation contaminants.The process solutions preferably are exposed to a degassing system forremoving oxygen. The MIPC separation particles are preferably alkylatednonporous polymer beads having an average diameter of about 1-100microns.

The method can further include contacting mobile phase solutions andsample solutions entering the column with multivalent cation captureresin before the solutions enter the column to protect the separationbed from multivalent cation contamination. The method is used toseparate single and double stranded DNA, and preferably used forseparating double-stranded polynucleotide fragments, particularly thosehaving 5 or more base pairs.

In an alternative method for improving the separation of polynucleotidefragments into fractions during MIPC using a liquid chromatographiccolumn containing a separation bed comprising MIPC separation particles,process solutions are contacted with multivalent cation capture resinbefore the solutions enter the chromatographic column in order toprotect the separation bed from multivalent cation contamination. Theseparation can be based on the fragment size or polarity. Themultivalent cation capture resin can be cation exchange resin orchelating resin, but is preferably a cation exchange resin having an ionexchange moiety selected from the group consisting of iminodiacetate,nitriloacetate, acetylacetone, arsenazo, hydroxypyridinone, and8-hydroxyquinoline groups. Cation exchange resin having animinodiacetate group is particularly preferred. The multivalent cationcapture resin is preferably contained in a guard disk, guard column, orguard cartridge. The MIPC separation particles are preferably nonporousbeads having an average diameter of about 1-100 microns. The particleshave been subjected to an acid wash treatment during manufacture inorder to eliminate multivalent cation contaminants. The method canfurther include supplying and feeding solutions entering the column withcomponents having process solution-contacting surfaces which arematerial which does not release multivalent cations into processsolutions so that the contents of the column are protected frommultivalent cation contamination. The process solution-contactingsurfaces of the components are preferably titanium, coated stainlesssteel, and organic polymer. The surfaces are subjected to a multivalentcation removal treatment. The treatment can include contacting thesurfaces with an aqueous solution containing nitric acid, phosphoricacid, pyrophosphoric acid, or chelating agents. In one embodiment, theprocess solutions include a mobile phase additive (e.g. EDTA or crownether) that can capture or bind multivalent cation contaminants. Theprocess solutions preferably are exposed to a degassing system forremoving oxygen. The method is preferably used for separatingdouble-stranded polynucleotide fragments, particularly those having 10or more base pairs.

Also disclosed herein is a method for improving separation ofpolynucleotide fragments during slalom chromatography using a liquidchromatographic column containing a separation bed comprising slalomchromatography DNA separation particles. The method comprises contactingthe process solutions with multivalent cation capture resin before thesolutions enter the chromatographic column in order to protect theseparation bed from multivalent cation contamination. The multivalentcation capture resin can be cation exchange resin or chelating resin,but is preferably cation exchange resin having an ion exchange moietyselected from the group consisting of iminodiacetate, nitriloacetate,acetylacetone, arsenazo, hydroxypyridinone, and 8-hydroxyquinolinegroups. Cation exchange resin having an iminodiacetate group isparticularly preferred. The multivalent cation capture resin ispreferably contained in a guard disk, guard column, or guard cartridge.

The method can further include supplying and feeding solutions enteringthe column with components having process solution-contacting surfaceswhich are material which does not release multivalent cations intoprocess solutions so that the contents of the column are protected frommultivalent cation contamination. The process solution-contactingsurfaces of the components are preferably titanium, coated stainlesssteel, or organic polymer. The surfaces are preferably subjected to amultivalent cation removal treatment. The treatment can includecontacting the surfaces with an aqueous solution containing nitric acid,phosphoric acid, pyrophosphoric acid, or chelating agents. In oneembodiment, the process solutions include a mobile phase additive (e.g.EDTA) that can capture or bind multivalent cation contaminants. Theprocess solutions preferably are exposed to a degassing system forremoving oxygen. The slalom chromatography method is preferably used forseparating double-stranded polynucleotide fragments, particularly thosehaving 5000 or more base pairs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatogram of double-stranded DNA separationillustrating the degradation effect on peak separation caused by ioniccontaminants present within the HPLC system. In this case, thedegradation effect is greater than about 174 base pairs.

FIG. 2 shows further contamination and degradation of the HPLCseparation now affecting all dsDNA fragments down to 80 base pairs, withthe larger fragments being affected the most.

FIG. 3 shows a chromatogram of dsDNA separation following injection ofthe column with tetrasodium EDTA, a metal chelating agent.

FIG. 4 shows a chromatogram of dsDNA separation obtained when a guarddisk containing gel-type iminodiacetate was positioned directly in frontof the sample injection valve of the HPLC system.

FIG. 5 shows a chromatogram of dsDNA separation obtained when a guarddisk containing gel-type iminodiacetate was placed directly in front ofthe separation column of the HPLC system (i.e., between the sampleinjection valve and the separation column).

FIG. 6A shows a guard disk having a one-piece annular ring.

FIG. 6B is an exploded view of a guard disk having a two-piece annularring and containing three pads of guard disk material (i.e., a layer orpad of multivalent cation capture resin which has been incorporated intoa fabric or membrane).

FIG. 6C shows an assembled view of the guard disk of FIG. 6B.

FIG. 7 shows placement of a chelating guard column and chelating guarddisk in a liquid chromatographic system for polynucleotide separation.

FIG. 8 shows placement of a chelating guard disk positioned between achromatographic separation column and a column top, where the guard diskis in direct contact with a titanium frit at the top portion of theseparation column.

FIG. 9 shows a high resolution MIPC separation of DNA restrictionfragments after the first injection of sample using a column havinguntreated PEEK frits.

FIG. 10 shows the chromatogram from the fifth injection of DNArestriction fragments using the column of FIG. 9.

FIG. 11 shows a separation performed as in FIG. 9 but using a columnhaving the same frits that were used in the column of FIG. 10 but whichhad been subjected to a nitric acid wash treatment.

FIG. 12 shows a separation performed as in FIG. 9 but using a columnhaving the same frits that were used in the column of FIG. 11 but whichhad been subjected to an acid wash treatments with HNO₃ and HCl.

FIG. 13 shows a separation performed as in FIG. 9 but using a columnhaving titanium frits.

FIG. 14 shows a high resolution MIPC separation of DNA restrictionfragments after the first injection of sample using a column havinguntreated PEEK frits.

FIG. 15 shows separation of a 20-mer oligonucleotide on a column havingtitanium frits.

FIG. 16 shows separation of the oligonucleotide as used in FIG. 15 buton a column having PEEK frits.

FIG. 17 shows separation of the oligonucleotide as used in FIG. 15 buton a column having PEEK frits which were treated with HNO₃ and HCl.

DETAILED DESCRIPTION OF THE INVENTION

Matched Ion Polynucleotide Chromatography (MIPC) as used herein, isdefined as a process for separating single and double strandedpolynucleotides using non-polar beads which have a pore size which iseffective to exclude the polynucleotides being separated, wherein theprocess uses counter ion agents, and uses an organic solvent to desorbthe polynucleotide from the beads.

The term polynucleotide is defined as a linear polymer containing anindefinite number of nucleotides, linked from one ribose (ordeoxyribose) to another via phosphoric residues. The present inventioncan be used in the separation of RNA or of double- or single-strandedDNA. For purposes of simplifying the description of the invention andnot by way of limitation, the separation of double-stranded DNA will bedescribed hereinafter, it being understood that all polynucleotides areintended to be included within the scope of this invention.

In parent application Ser. No. 08/748,376, filed Nov. 13, 1996, and inU.S. Pat. No. 5,585,236, the DNA separation process was characterized asreverse phase ion pair chromatography (RPIPC). However, since RPIPC doesnot incorporate certain essential characteristics described in thepresent invention, another term, MIPC, has been selected for this DNAseparation process.

The system of the invention includes a chromatographic column containinga separation bed of MIPC separation particles held in the column betweenporous frits positioned at each end of the column. The term “MIPCseparation particles” refers to any material which is capable ofseparating polynucleotide fragments by MIPC. The MIPC separationparticles can be inorganic, including silica, zirconia, alumina, orother material; or can be polymeric, including crosslinked resins ofpolystyrene, polyacrylates, polyethylene, or other organic polymericmaterial. The only requirement for the MIPC separation particles is thatthey must have a surface that is either intrinsically hydrophobic or bebonded with a material that forms a surface having sufficienthydrophobicity to interact with a counter ion agent. Suitable MIPCseparation particles are described in co-pending U.S. patent applicationSer. Nos. 09/058,580 and 09/058,337 which are hereby incorporated byreference in their entirety.

As used herein, the term “nonporous” is defined to denote a bead whichhas surface pores having a diameter that is less than the size and shapeof the smallest DNA fragment in the separation in the solvent mediumused therein. Included in this definition are polymer beads having thesespecified maximum size restrictions in their natural state or which havebeen treated to reduce their pore size to meet the maximum effectivepore size required.

The surface conformations of nonporous beads of the present inventioncan include depressions and shallow pit-like structures which do notinterfere with the separation process. A pretreatment of a porous beadto render it nonporous can be effected with any material which will fillthe pores in the bead structure and which does not significantlyinterfere with the MIPC process.

Pores are open structures through which mobile phase and other materialscan enter the bead structure. Pores are often interconnected so thatfluid entering one pore can exit from another pore. We believe thatpores having dimensions that allow movement of the polynucleotides intothe bead structure result in poorly resolved separations or separationsthat have very long retention times. In MIPC, however, the beads are“nonporous” and the polynucleotides do not enter the bead structure.

Suitable MIPC separation particles comprise alkylated nonporous polymerbeads having an average diameter of about 1-100 microns, which aredescribed in further detail in the “Methods” section below.

Other components of the liquid chromatography system include aninjection valve and one or more mobile phase supply means. Mobile phasesupply means is (are) connected to the injection valve, and theinjection valve is connected to the inlet of the chromatographicseparation column, by means of conduit (e.g., tubing), as illustrated inFIG. 8.

The components of the liquid chromatography system have surfaces (i.e.,“process solution-contacting surfaces”) which contact process solutionsheld within the component (e.g., the mobile phase supply means) orflowing through the component (e.g., the porous frits, chromatographiccolumn, injection valve, and conduits). The term “process solution” asused herein refers to any solution (such as a sample solution or mobilephase solution) which is contained within or flows through any componentof the liquid chromatography system during liquid chromatography. Theterm “process solution-contacting surface” refers to any surface of aliquid chromatography system to which process solutions are exposedduring performance of chromatographic separations.

The process solution-contacting surfaces of the porous frits on eitherend of the separation column must be made of material which does notrelease multivalent cations into aqueous solutions flowing through thecolumn. The material is preferably titanium, coated stainless steel, ororganic polymer, but is most preferably acid treated titanium asdescribed hereinbelow. The term “coated stainless steel” as used hereinrefers to stainless steel that has been coated so that does not release,or is prevented from releasing, multivalent cations. A non-limitingexample of a coating material is polytetrafluoroethylene (i.e.,Teflon®). “Coated stainless steel” as used herein also refers tostainless steel that has been pre-treated with an agent such as EDTA orphosphoric acid which forms coordination complexes with multivalentmetal ions.

“Passivated stainless steel” as used herein refers to stainless steelthat has been treated with an agent that removes the oxidized metals andalso metals that are easily oxidized such as iron. The most commonpassivating agent for stainless steel is nitric acid. Nitric acid willremoved any oxidized metals, but will also remove iron that is locatedon the surface of the metal, leaving other metals such as chromium andnickel. Some chelating agents can coat and passivate. EDTA will firstcoat oxidized metals especially colloidal iron oxide particles. Astreatment continues, the EDTA will bind and dissolve the iron oxide.However, as individual iron molecules leave the particle, otherchelating molecules must coat the newly exposed surfaces for the surfaceto remain suitable for MIPC. A chelating agent does not passivate in thesense that it will only coat metal ions for which it is specific andwill not dissolve non-oxidized metals. The chelating reagents useddepend upon the type of ion contamination which is present. For example,Tiron chelating agent is selective for titanium and iron oxides. EDTA isselective for most metal oxides at pH 7. Other chelating reagentsinclude cupferron, 8 hydroxyquinoline, oxine, and various iminodiaceticacid derivatives. If the chelating reagents are to be used aspassivating reagents as well as coating reagents, then it is importantthe metal ion chelate complex, for example, EDTA-metal ion complex, issoluble in the fluid. Chelating reagents that form insoluble complexes,for example 8-hydroxyquinoline, perform coating functions only.

Without wishing to be bound by theory, it is believed that oxidized andpositively charged metals, such as oxides of iron on the surface ofstainless steel can trap negatively charged molecules such as DNAleading to degradation of the chromatographic separation, and that thepre-treatment masks or shields these surface charges. EDTA can be added,for example, in an amount sufficient to shield any surface sites whichwould interfere with the chromatographic separation. In one embodiment,a solution of a metal chelating agent such as EDTA can be applied in abatch process to coat the surface, for example by a single injection ofEDTA solution into the HPLC system. In another embodiment, EDTA isincluded as an additive in the mobile phase.

Other components of the liquid chromatography system are preferablyketone (PEEK) or polyethylene. The preferred system tubing (i.e.,conduit) is titanium, PEEK, or other polymeric material, with an innerdiameter of 0.007″. The preferred mobile phase inlet filters arecomposed of a porous, non-stainless steel material, which can be PEEK,polyethylene, or other polymeric material. The preferred solvent pump isalso made of a non-stainless steel material; the pump heads, checkvalves, and solvent filters are preferably titanium, PEEK, or otherpolymeric material. The preferred degasser is an inline degasser placedprior to the pump inlet. The sample injection valve is also preferablytitanium, PEEK, or other polymeric material. A standard detector andmobile phase reservoirs can be used, with no modifications necessary.

Materials such as titanium, PEEK and other organic polymers such aspolyethylene, have been generally considered to be inert and preferredfor the separation of biological molecules by processes in use beforethe development of MIPC. We have discovered that these materials, whileinert for the prior art processes, can be a source of contaminants whichinterfere with the MIPC chromatographic separation. We have alsoobserved that the interference with MIPC separation by these materialsbecomes more apparent during separations carried out at elevatedtemperatures, e.g. 57° C. as compared to 51° C.

In a preferred embodiment of the present invention, all of the processsolution-contacting surfaces are subjected to a multivalent cationremoval treatment to remove any potential source of multivalent cationcontamination. These surfaces include the column inner surface, porousfrits, conduits, mobile phase supply system, injector valves, mixers,pumpheads, and fittings. A non-limiting example of a multivalent cationremoval treatment is an acid wash treatment. This wash treatment caninclude flushing or soaking and can include sonication. An example of anacid wash treatment is sonication of a PEEK or titanium frit in thepresence of aqueous nitric acid solution, followed by sonication inwater until a neutral pH is achieved. Other treatments includecontacting the surfaces with chelating agents such as EDTA,pyrophosphoric acid, or phosphoric acid (e.g. 30% by weight phosphoricacid).

PEEK and titanium can be treated with dilute acids including nitric andhydrochloric acids. PEEK is not compatible with concentrated sulfuric orconcentrated nitric acids. Titanium is not compatible with concentratedhot hydrochloric acid. Treatment with a chelating reagent can beperformed before, but preferably after treatment with an acid. 20 mMtetrasodium EDTA is a preferred chelating reagent treatment.

The preferred treatment for titanium frits is sonication for 10 minuteswith cold hydrochloric acid, sonication with water until neutral pH, 2hour sonication with 0.5 M tetrasodium EDTA, storage several days in 0.5M tetrasodium EDTA, sonication with water until neutral pH, and thenwashing with methanol, followed by drying. Preferred treatment for PEEKfrits is sonication for 15-30 minutes each with THF, concentratedhydrochloric acid, 20% nitric acid, sonication with water until neutralpH, and then washing with methanol, followed by drying. Although this isa preferred treatment method, the effectiveness of this treatment ofPEEK frits can depend on the vendor and lot of material treated. Thesuccess of the treatment also depends on the temperature of the DNAseparation with higher column temperatures requiring the most completeremoval of contamination. Although the mechanism is unknown, the effectof PEEK contamination is dependent on the sequence of the fragment (seeExamples hereinbelow).

If the ionic contaminant is organic, then organic solvents or acombination of organic solvents and acids can be used. Also, organicionic contaminants can require detergents, soaps or surfactants forremoval from the surface. Nonionic contaminants such as greases and oilswill also contaminate the separation column, generally leading to poorpeak shape, but depending upon the size of the fragment. Nonionicorganic contaminants such as oils will require detergents, soaps orsurfactants to remove. Column tubing can be treated under sonicationwith Decalin (D5039, Sigma) to remove silicon greases and oils. Removalof colloidal metal oxides such as colloidal iron oxide can requirerepeated or continuous treatment as the surface of the particle isdissolved and new metal oxides are exposed.

The preferred embodiment of the liquid chromatography system of thepresent invention utilizes methods to minimize the exposure of allprocess solution-contacting surfaces to oxygen. Dissolved oxygen withinthe mobile phase, for example, can react with exposed metals on thesesurfaces to form oxides which will interfere with the MIPCchromatographic separation. The liquid chromatography system preferablyemploys a degassing method for essentially removing dissolved oxygenfrom the mobile phase prior to contact with the rest of thechromatography system. Examples of degassing methods include sparging ofthe mobile phase with an inert gas such as argon or helium, or filteringthe mobile phase under vacuum. A preferred method uses a vacuum typedegasser which employs inline passage of the mobile phase over one sideof an oxygen permeable membrane system where the other side is subjectedto a vacuum. An example of a suitable four channel vacuum type degasseris Degaset™, Model 6324 (MetaChem Technologies, Torrance, Calif.).

In another embodiment of the invention, a stainless steel HPLC systemcan be used if a component for removing multivalent cations, hereinreferred to as a “multivalent cation capture resin,” is also used. Amultivalent cation capture resin is preferably a cation exchange resinor chelating resin. Any suitable cation exchange resin or chelatingresin can be used. Preferred cation exchange and chelating resins aredescribed below.

Cation exchange resins having an ion exchange moiety selected from thegroup consisting of iminodiacetate, nitriloacetate, acetylacetone,arsenazo, hydroxypyridinone, and 8-hydroxyquinoline groups areparticularly preferred. Cation exchange resins having hydroxypyridinonegroups are especially useful for removing iron from the system. Cationexchange resins having iminodiacetate groups are particularly preferredfor use in the present invention because of their wide availability inresin format.

A chelating (i.e., coordination binding) resin is an organic compoundwhich is capable of forming more than one non-covalent bond with ametal. Chelating resins include iminodiacetate and crown ethers. Crownethers are cyclic oligomers of ethylene oxide which are able to interactstrongly with alkali or alkaline earth cations and certain transitionmetal cations. A cavity in the center of the molecule is lined withoxygen atoms which hold cations by electrostatic attraction. Each crownether has a strong preference for cations whose ionic radius best fitsthe cavity.

The multivalent cation capture resin is preferably contained in a guardcolumn, guard cartridge, or guard disk. Guard columns and cartridges arefrequently used to protect liquid chromatography columns fromcontamination and are widely available. In their normal use, guardcolumns and cartridges typically contain packing material which issimilar to the stationary phase of the separation column. However, foruse in the present invention, the guard column or cartridge must containa multivalent cation capture resin. The guard disc or guard column mustcontain beads in which the metal ions can be trapped, but where DNAcannot enter the bead and be trapped.

For use in the system of the present invention, the guard cartridge or 1column should be sufficiently large to provide adequate capacity, butmust be small enough to allow effective gradient elution to be used. Apreferred guard cartridge has a void volume of less than 5 mL, morepreferably, less than 1 mL, so that the mobile phase gradient is notdelayed by more than 5 minutes and, preferably, less than 1 minute. Thepreferred cartridge has a 10×3.2 mm bed volume.

Guard disks are described in detail in U.S. Pat. No. 5,338,448, which isincorporated herein by reference in its entirety. For use in the presentinvention, a guard disk comprises a layer or pad of a multivalent cationcapture resin which has been incorporated into a fabric or membrane sothat the resin is not separable from the guard disk under liquid flowconditions present during the performance of chromatographicseparations.

In its preferred form, the guard disk is circular, having a rigidannular outer ring or collar for easy handling. The annular ring can beconstructed of any suitable material which is inert to thechromatographic separation, such as inert conventional engineeringplastic. The only requirement for the material is that it must be inertto the mobile phase and sample and have sufficient dimensionalstability. The rigid annular outer ring of the guard disk can comprise asingle rigid annular outer ring encircling a disk-shaped pad of guarddisk material. As used herein, the term “guard disk material” refers toa layer or pad of multivalent cation capture resin which has beenincorporated into a fabric or membrane.

As shown in FIG. 6A, one or more pads of guard disk material 2 areplaced in the rigid annular ring 4. For example, the fabric can be cutto a circular diameter which securely contacts the inner diametersurface of the annular ring. As the disk holder is tightened against thedisk, the top and bottom surfaces of the holder seal against the collarof the guard disk. Sealing pressure from the guard disk holder is,therefore, applied against the collar of the disk which prevents thematerial of the guard disk pad from being crushed.

Alternatively, the rigid annular outer ring can comprise two flangedrings, as shown in FIGS. 6B and 6C, an outer flanged ring 6 and an innerflanged ring 8, where the inner flanged ring is insertable within theflange of the outer ring, forming a press-fit two-piece collar aroundone or more pads of guard disk material 10. Preferably, the innerdiameter (a) of the inner flanged ring will have the same diameter asthe separation column bed.

In the two-piece annular ring embodiment shown in FIG. 6C, one or morepads of guard disk material 10 having a diameter greater than the innerdiameter (b) of the outer flanged ring 6 are positioned within theflanges of the outer ring. The inner flanged ring 8 is then insertedinto the outer ring to form a press-fit two-piece annular ring in whichthe guard disk pad(s) is (are) frictionally held within the press-fitring or collar. Preferably, the inner diameter (b) of the outer flangedring and the inner diameter (a) of the inner flanged ring aresubstantially the same.

Alternatively, the rigid annular outer ring can be incorporated into theguard disk holder or chromatographic column cap. The annular ring is aflange that is part of one or both sides of the disk holder or thecolumn cap. In this case, the guard disk does not have an outer ring. Acircle of the guard disk sheet material is placed into the holder orcolumn cap. The flange in the holder column cap is annular so that, whenthe holder or column cap is tightened, the flange pinches or seals theouter annular portion of the guard disk. The center portion of the guarddisk not pinched is in a chamber or depression in the holder or cap.Fluid flows through the center portion, allowing the guard disk toretain particulate or strongly adsorbed material, but fluid cannot flowaround the disk or past the edges. The function of the guard disk isexactly the same as when the collar is part of the guard disk itself.However, in this case, the collar is part of the holder or column cap.

In the system of the invention, a multivalent cation capture resincontained in a guard column, guard cartridge, or guard disk is placedupstream of the separation column. Preferably, the guard column,cartridge, or disk containing the resin is placed upstream of the sampleinjection valve. Although this is preferably a guard disk, a guardcartridge or column can be used as long as the dead volume of thecartridge or column is not excessive and an effective mobile phasegradient can be produced.

Additionally, a second guard disk, column, or cartridge can be placedbetween the sample injection valve and the separation column. In certaincases, the second guard disk (or cartridge or column) can be avoided ifthe contaminants are sufficiently cleaned by a guard column placedupstream of the injection valve, or if the contaminants are avoidedthrough the use of nonmetal or titanium components throughout the HPLCsystem.

Placement of a chelating guard column and chelating guard disk in aliquid chromatography system for polynucleotide separation isillustrated in FIG. 7. The mobile phase reservoirs 12 contain mobilephase inlet filters 14 which are connected to the solvent pump 16 bysystem tubing 18. The solvent pump 16 is connected to a chelating column20 by system tubing 18. The chelating column 20 is connected to thesample injection valve 22 by system tubing 18. The sample injectionvalve has means for injecting a sample (not shown). The sample injectionvalve 22 is connected to a chelating guard disk 24 by system tubing 18.The chelating guard disk 24 is connected to the inlet (not shown) of theseparation column 26 by system tubing 18. Detector 28 is connected tothe separation column 26. As discussed above, the system tubing, mobilephase inlet filters, solvent pump, sample injection valve, andseparation column are preferably made of titanium, coated stainlesssteel, or organic polymer. The material is preferably treated so that itdoes not release multivalent cations. The treatment can includetreatment with nitric acid, phosphoric acid, pyrophosphoric acid, orchelating agents. In cases, where components of the HPLC do not releasemetal ion contaminants and are suitable for MIPC, then use of thechelating cation exchange guard column or guard disc is not necessary.

In operation, mobile phase from the mobile phase reservoirs 12 is pumpedthrough mobile phase inlet filters 14 by solvent pump 16. By way ofsystem tubing 18, the mobile phase stream flows through chelating column20, through sample injection valve 22, through chelating guard disk 24,then into separation column 26. Detector 28 is located downstream fromseparation column 26.

FIG. 8 illustrates a specific embodiment of the invention in which achelating guard disk is placed in direct contact with a titanium frit atthe top portion of a chromatographic separation column. Column top 30has conventional fittings for receiving mobile phase and sample throughinlet tubing 32. The column top or cap 30 is fitted and sealablyattached to column body 34 containing chromatographic bed 36 using aconventional fitting 38 (e.g., threaded) or any equivalent fittingcapable of tightly sealing the column top to the column body. The columntop 30 is adapted to receive the chelating guard disk 40 in a sealingcavity 42. In this embodiment, the guard disk 40 is in direct contactwith a titanium column frit 44, which is located at the upstream end ofthe column body 34 to prevent disturbance of the chromatographic bed 36when the column top 30 is removed to observe the guard disk.

In operation, solvent pump 46 pumps elution solvent to sample injectionvalve 48 into column top 30 through chelating guard disk 40 and thenthrough titanium frit 44 before entering chromatographic bed 36. Mobilephase pressure upstream from the guard disk is measured by pressuretransducer 50 which is electrically connected to a display device 52.

As discussed above, a chelating guard column, cartridge, or disk can beused in conjunction with a conventional, stainless steel liquidchromatography system, or with a system containing non-metal or titaniumcomponents in order to provide extra protection against ioniccontaminants. For additional column protection, a mobile phasecontaining 0.1 mM tetrasodium EDTA or other chelating solution can beused during the performance of polynucleotide separations.

The methods of the invention comprise using the improved systemsdescribed above to separate mixtures of polynucleotide fragments,particularly double-stranded polynucleotide fragments. The methods ofthe present invention can be used to separate polynucleotide fragmentshaving up to about 1500 base pairs using MIPC and up to about 20,000base pairs using slalom chromatography.

In most cases, the method will be used to separate polynucleotideshaving 80 or more base pairs, up to about 1500 base pairs. The methodprovides good separation and reliability for longer polynucleotideshaving base pairs within the range of 10-100, but is also useful forbase pairs less than 10.

The polynucleotides which can be separated by the present method includedouble-stranded polynucleotides. Since the mechanism of the degradationeffect is still unknown, it is not known how significantlysingle-stranded polynucleotide separations are affected. Furthermore,only short (25-mer) single-stranded polynucleotides are usuallyseparated by liquid chromatographic methods. With short lengths, theeffect is more difficult to detect.

Samples containing mixtures of polynucleotides can result from totalsynthesis of polynucleotides, cleavage of DNA with restrictionendonucleases or RNA, as well as polynucleotide samples which have beenmultiplied or amplified using polymerase chain reaction (PCR) techniquesor other amplifying techniques. Also, the separations of polynucleotidescan be performed at different temperatures. As the temperature of thecolumn is increased, heteroduplexes that may be present in the sampleswill partially melt and elute earlier than homoduplexes in the sample.The separation is carried out under conditions effective to at leastpartially denature the heteroduplexes (e.g. thermal or chemicaldenaturing) resulting in the separation of the heteroduplexes from thehomoduplexes. Under these conditions, the homoduplexes are eluted undersize dependent conditions, or at higher temperatures, size and sequencedependent conditions, and the heteroduplexes are separated under sizeand sequence dependent conditions (P. Underhill, Proc. Natl. Acad. Sci.93:196-200 (1996)).

The systems of the present invention are preferably used to separatedouble-stranded polynucleotide fragments by MIPC. The preferred methodis described by Bonn et al. in U.S. Pat. No. 5,585,236, which isincorporated herein by reference in its entirety. The method of Bonn etal. utilizes separation columns filled with nonporous polymeric beadshaving an average diameter of about 1-100 microns, preferably 1-10microns, more preferably 1-5 microns. Beads having an average diameterof 1.5-3.0 microns are most preferred.

The nonporous polymeric beads are prepared by a two-step process inwhich small seed beads are initially produced by emulsion polymerizationof suitable polymerizable monomers. The emulsion polymerizationprocedure of the invention is a modification of the procedure of J. W.Goodwin et al. (Colloid & Polymer Sci., 252:464-471 (1974)). Monomerswhich can be used in the emulsion polymerization process to produce theseed beads include styrene, alkyl substituted styrenes, alpha-methylstyrene, and alkyl substituted alpha-methyl styrenes, preferablymonomers where the benzene-type ring is substituted with 1-4 C₁₋₆ alkylgroups, and the monomers described, for example, in U.S. Pat. No.4,563,510. The seed polymer beads are then enlarged and alkylated, asdescribed by Bonn et al. in U.S. Pat. No. 5,585,236.

In MIPC, the polynucleotides are matched with a counter ion agent andthen subjected to chromatography using the alkylated beads describedabove. The identity of the counter ion agent can be varied andconventional agents capable of forming ion pairs with polynucleotidescan be used. Typical counter ion agents include trialkylammonium saltsof organic or inorganic acids, for example, trimethyl, triethyl,tripropyl, and tributyl ammonium acetates, halides, etc. A particularlypreferred counter ion agent is triethylammonium acetate (TEAA).Tetraalkylammonium salts have also been used such as (25 mM)tetrabutylammonium bromide.

To achieve high resolution chromatographic separations ofpolynucleotides, it is generally necessary to tightly pack thechromatographic column with the solid phase polymer beads. Any knownmethod of packing the column with a column packing material can be usedto obtain adequate high resolution separations. Typically, a slurry ofthe alkylated polymer beads is prepared using a solvent having a densityequal to or less than the density of the polymer beads. The column isthen filled with the polymer bead slurry and vibrated or agitated toimprove the packing density of the polymer beads in the column.Mechanical vibration or sonication are typically used to improve packingdensity.

For example, to pack a column having an inner diameter of 50×4.6 mL, 1.4grams of alkylated beads are suspended in 15 mL of tetrahydrofuran withthe help of sonication. The suspension is then packed into the columnusing 50 mL of methanol at 70 MPa of pressure. In the final step, thepacked bed is washed with 50 mL of deionized water. This reduces theswelling of the beads and improves the density of the packed bed.

Alternatively, slalom chromatography can be used to separate larger DNAfragments (i.e., 5000 or more base pairs) according to the methods ofthe invention. Slalom chromatography, as described by J. Hirabayashi etal. (Anal. Biochem., 178:336-341 (1989); Biochemistry, 29:9515-9521(1990)), is a method of separating DNA fragments having dimensionscomparable to the chromatographic particles. In practice, this meansthat currently available columns separate fragments in the range of5000-50,000 base pairs. Fragments are eluted in order of size, with thesmallest fragments eluting first, opposite to the order of gelpermeation. The mechanism is believed to be hydrodynamic sieving, ratherthan surface interactions between the DNA and the chromatographicpacking. Particle size and mobile phase flow rate have the greatestinfluence on separation. While the mobile phase is usually aqueousbuffer, organic or aqueous organic mobile phases are not excluded.

In slalom chromatography, the chromatographic separation column ispacked with “slalom chromatography DNA separation particles”. The term“slalom chromatography DNA separation particles” refers to any materialwhich is capable of separating DNA fragments by slalom chromatography.Slalom chromatography separation particles can be inert organicpolymers, inert inorganic polymers, silica, or cation exchange resin.The only requirement for the slalom chromatography DNA separationparticles is that they must have little interaction with the DNAfragments.

Procedures described in the past tense in the examples below have beencarried out in the laboratory. Procedures described in the present tensehave not been carried out in the laboratory, and are constructivelyreduced to practice with the filing of this application.

EXAMPLE 1

Sodium chloride (0.236 g) was added to 354 mL of deionized water in areactor having a volume of 1.0 liter. The reactor was equipped with amechanical stirrer, reflux condenser, and a gas introduction tube. Thedissolution of the sodium chloride was carried out under inertatmosphere (argon), assisted by stirring (350 rpm), and at an elevatedtemperature (87° C.). Freshly distilled styrene (33.7 g) and 0.2184 g ofpotassium peroxodisulfate (K₂S₂O₈) dissolved in 50 mL of deionized waterwere then added. Immediately after these additions, the gas introductiontube was pulled out of the solution and positioned above the liquidsurface. The reaction mixture was subsequently stirred for 6.5 hours at87° C. After this, the contents of the reactor were cooled down toambient temperature and diluted to a volume yielding a concentration of54.6 g of polymerized styrene in 1000 mL volume of suspension resultingfrom the first step. The amount of polymerized styrene in 1000 mL wascalculated to include the quantity of the polymer still sticking to themechanical stirrer (approximately 5-10 g). The diameter of the sphericalbeads in the suspension was determined by light microscopy to be about1.0 micron.

Beads resulting from the first step are still generally too small andtoo soft (low pressure stability) for use as chromatographic packings.The softness of these beads is caused by an insufficient degree ofcrosslinking. In a second step, the beads are enlarged and the degree ofcrosslinking is increased. The protocol for the second step is based onthe activated swelling method described by Ugelstad et al. (Adv. ColloidInterface Sci., 13:101-140 (1980)). In order to initiate activatedswelling, or the second synthetic step, the aqueous suspension ofpolystyrene seeds (200 mL) from the first step was mixed first with 60mL of acetone and then with 60 mL of a 1-chlorododecane emulsion. Toprepare the emulsion, 0.206 g of sodium dodecylsulfate, 49.5 mL ofdeionized water, and 10.5 mL of 1-chlorododecane were brought togetherand the resulting mixture was kept at 0° C. for 4 hours and mixed bysonication during the entire time period until a fine emulsion of <0.3microns was obtained. The mixture of polystyrene seeds, acetone, and1-chlorododecane emulsion was stirred for about 12 hours at roomtemperature, during which time the swelling of the beads occurred.Subsequently, the acetone was removed by a 30 minute distillation at 80°C. Following the removal of acetone, the swollen beads were furthergrown by the addition of 310 g of a ethyidivinylbenzene anddivinylbenzene (DVB) (1:1.71) mixture also containing 2.5 g ofdibenzoylperoxide as an initiator. The growing occurred with stirringand with occasional particle size measurements by means of lightmicroscopy.

After completion of the swelling and growing stages, the reactionmixture was transferred into a separation funnel. In an unstirredsolution, the excess amount of the monomer separated from the layercontaining the suspension of the polymeric beads and could thus beeasily removed. The remaining suspension of beads was returned to thereactor and subjected to a stepwise increase in temperature (63° C. forabout 7 hours, 73° C. for about 2 hours, and 83° C. for about 12 hours),leading to further increases in the degree of polymerization (>500). Thepore size of beads prepared in this manner was below the detection limitof mercury porosimetry (<30 Å).

After drying, the dried beads (10 g) from step two were suspended in 100mL of 1-chlorododecane and stirred (370 rpm) for 12 hours at 100° C.following addition of 1 g of aluminum chloride. At the end of thisperiod, the reaction mixture was cooled to 80° C. and mixed with 150 mLof 4M hydrochloric acid. After 2 minutes of stirring, the reactionmixture, now containing hydrochloric acid, was transferred into aseparation funnel and overlaid by 300 mL of n-heptane. The phases werestirred into each other and, after subsequent separation of phases, theaqueous phase was removed and discarded. The remaining organic phase waswashed two additional times with 200 mL of 1M hydrochloric acid andsubsequently centrifuged at 5000 rpm. The separated beads were washedfour times with 100 mL of n-heptane, and then two times with each of thefollowing: 100 mL of diethylether, 100 mL of dioxane, and 100 mL ofmethanol. Finally, the beads were dried.

Alternatively, the alkylation was carried out using tin chloride bymeans of a procedure which is otherwise similar to that utilizingaluminum chloride. One hundred mL (100 mL) of 1-chlorooctadecane, 10 gof poly(styrene/ethylstyrene/divinylbenzene) beads, and 5 mL of SnCl₄were stirred at 100° C. for 12 hours. The mixture was cooled to roomtemperature, 100 mL of n-heptane was added and the mixture was thenextracted with 4×300 mL of water in a separation funnel. Subsequentcentrifugation was carried out for 5 minutes at 5000 rpm. Thesupernatant and 1-chlorooctadecane were discarded and water was removedas completely as possible. Washing with 2×150 mL of n-heptane, 2×150 mLof dioxane, and 2×150 mL of methanol completed the procedure. Each ofthe washing steps was followed by centrifugation at 5000 rpm. Thealkylated beads were then dried at 60° C.

Alkylation of the aromatic rings of the polymer was verified by FourierTransform Infrared spectroscopy (FTIR). The beads differed only slightlyin size from each other. The mean value for the particle diameter wasfound to be 2.10 microns, with a standard deviation of 0.12 micron.

The beads preferably are subjected to an acid wash treatment in order toessentially eliminate multivalent cation contaminants. The beadsprepared are washed three times with tetrahydrofuran and two times withmethanol. Finally the beads are stirred in a mixture containing 100 mLtetrahydrofuran and 100 mL concentrated hydrochloric acid for 12 hours.After this acid treatment, the polymer beads are washed with atetrahydrofuran/water mixture until neutral (pH=7). The beads are thendried at 40° C. for 12 hours.

The separation of single- and double-stranded DNA was accomplished usingMIPC. Triethylammonium acetate was used as the counter ion agent.Elution was effected with the help of a linear organic solvent gradientof acetonitrile. The chromatographic conditions were as follows: Column:50×4.6 cm i.d. Mobile phase: 0.1 M TEAA, pH 7.0. Gradient: 7.5-13.75%acetonitrile in 4 minutes, followed by 13.75-16.25% acetonitrile in 6minutes. Flow rate: 1 mL/min. Column temperature: 50° C. Detection: UVat 254 nm. Sample: 0.5 μg pBR322 DNA-Hae III restriction digest.

EXAMPLE 2

Seed polystyrene latex was prepared using 0.374 g of NaCl, 0.1638 g ofK₂S₂O₈, 404 mL of water, and 37 mL of styrene, stirred at 81° C. at 350rpm for 6 hours. The resulting seed particles had a diameter of 1.3microns. Then, 200 mL of the seed latex was swollen with a mixture of 50mL of divinylbenzene, 0.5 mL of dibenzoylperoxide, and 5 mL of acetone.The mixture was stirred for 6 hours at 40° C. and 1 hour at 45° C. Thefinal diameter of the particles was 1.8 microns. Next, the excessdivinylbenzene was removed and the particles polymerized for 12 hours at65° C., followed by 14 hours at 85° C. After drying, alkylating, andcleaning, the polymer was used as described in Example 1.

EXAMPLE 3

Many experiments in molecular biology, including hybridization and DNAsequencing, require tagging of DNA with either radioactive isotopes,such as ³²P or ³³P or fluorescent dyes, such as fluorescein,2′,7′-dimethyoxy-4′,5′-dichlorofluorescein, tetramethylrhodamine, andrhodoamine, as described by S. Fung et al. (U.S. Pat. No. 4,855,225).Since the covalent coupling of radioisotopes and fluorescent dyes isusually incomplete, labeled DNA must be purified away from unreactedDNA, which otherwise will compete with the dye-labeled primers andprobes in sequencing and hybridization reactions.

The purification of labeled samples was accomplished simply and rapidlyby means of MIPC on nonporous, alkylated (C₁₈)poly(ethylvinylbenzene-divinylbenzene) beads. Recovery of DNA was atleast 96%.

The separation of fluorescent dye-labeled DNA from unreacted DNA canalso be achieved by reverse phase chromatography only, i.e., in theabsence of a counter ion reagent, because the hydrophobic nature of thefluorophore significantly increases the retention of the DNA on analkylated stationary phase relative to unreacted DNA. This is an exampleof separation based on polarity of the fragments.

EXAMPLE 4

If the gradient delay volume is minimized, the separation of PCRproducts and hybrid DNA derived from various sources of DNA, includingliving and dead organisms (animal and plant), as well as parts of suchorganisms (e.g., blood cells biopsies, sperm, etc.), onoctadecyl-modified, nonporous poly-(ethylvinylbenzene-divinylbenzene)beads can be achieved with run times under 2 minutes.

The analysis of PCR products and hybrid DNA usually requires onlyseparation and detection of one or two species of known length. Becauseof this, the resolution requirements are considerably less severe thanfor separations of DNA restriction fragments. Such less stringentresolution requirements allow the utilization of steep gradients and,consequently, lead to still shorter run times. The recovery rate for apolynucleotide fragment containing 404 base pairs was about 97.5%.

Unlike capillary electrophoresis (CE), PCR samples do not have to bedesalted prior to analysis by MIPC. This represents a decisive advantageof MIPC over CE. With MIPC, it is thus possible to achieve a fullyautomated analysis of PCR samples if an automatic autosampler isutilized. Moreover, since the volume of sample injection is known, incontrast to CE, quantitation over several orders of magnitude can beachieved without the need for an internal standard, hence allowing thequantitation of gene expression, as well as the determination of virustiters in tissues and body fluids. A fully automated version of themethod of the invention has been used to discriminate (i.e.,distinguish) normal from mutated genes, as well as to detect oncogenesand bacterial and viral genome DNA (hepatitis C virus, HIV,tuberculosis) for diagnostic purposes. Moreover, adjustment of columntemperature allows the stringency of hybridization reactions to bemodulated.

PCR methods and processes have been described by R. K. Sreke et al.(Science, 230:1350-1354 (1985)) and K. B. Mullis (U.S. Pat. No.4,683,202). These references are incorporated herein by reference for amore complete description of methods and processes for obtaining PCRsamples which can be separated using the method of the presentinvention.

EXAMPLE 5

A Hewlett-Packard HP1090 instrument formerly used for proteinseparations was purchased and outfitted with a column and mobile phasesas described in Examples 1 and 2. In this case, the column contained 0.5μm 316 stainless steel frits. The HP1090 instrument is available only asa stainless steel instrument.

The separation of a DNA standard resulted in inconsistently degradedperformance, as shown in FIGS. 1 and 2. Then, 0.1 mM tetrasodium EDTAwas added to mobile phases A and B. While this improved performance,degradation of the column still occurred after one day of use.

The instrument was then outfitted with a column containing 0.5 μmtitanium frits and an iminodiacetate guard disk positioned in front ofthe column. This modification resulted in several days of separationwith no degradation of performance.

Later, a guard cartridge with dimensions of 10×3.2 mm, containingiminodiacetate chelating resin of 2.5 mequiv/g capacity and 10 μmparticle size was positioned directly in front of the injection valve.This also resulted in elimination of the contamination problem. Thesystem was able to operate with or without the guard disk in this mode.

The minimum modifications needed to practice the invention to achieveimproved polynucleotide separations involve using a stainless steel HPLCsystem that has been retrofitted with a chelating disk or cartridgepositioned in front of the injection valve or column and a columncontaining titanium or PEEK frits. Newer HPLC equipment might be able tooperate with only changing the critical frits to titanium or PEEK andthen preventing further corrosion of the instrument with degassing ofthe mobile phase.

While the use of tetrasodium EDTA by itself is not always adequate, itsuse can degrade performance if the concentration is high and the EDTAcontributes to the driving ion action in the MIPC separation. EDTA canalso interfere with detection by mass spectrometry, or can interferewith the subsequent analysis of collected fractions.

The performance of any column capable of separating DNA can be improvedthrough the column protection methods and system described in thisexample. Other columns that have been used to separate double-strandedDNA include nonporous alkylated reverse phase silica materials, largepore poly(styrene/divinylbenzene) and other polymeric materials, andlarge pore silica-based reverse phase materials.

EXAMPLE 6

The column material described in Example 5 is packed in column bodyhardware consisting of titanium, PEEK, or other polymeric material, andthe column frits are titanium, PEEK, or other polymeric material. TheHPLC system still requires minimum column protection, as described inExample 5, consisting of a chelating ion exchange resin positioned infront of the injection valve or in front of the column.

EXAMPLE 7

The performance of the HPLC system was improved by employing polymericor titanium in all of the frits. In the case of a Hewlett-Packard HP1090instrument, polymeric inlet mobile phase filters replaced the standardstainless steel filters. Titanium frits replaced the stainless steelfrits used in the mixer after the pump head, but before the chelatingion exchange cartridge before the injector. This resulted in aseveral-week lifetime of the cartridges before breakthrough ofcontaminants occurred.

EXAMPLE 8

Alternatively, a PEEK or titanium HPLC system can be employed fordouble-stranded DNA separation. In this case, a column containingtitanium frits or PEEK frits must be used. Column protection consistingof the chelating cartridge positioned before the injection valve and/orthe guard disk positioned before the column is preferred, but notnecessary. Mobile phase containing a chelating reagent is preferred, butnot necessary, and is used only in cases where collection of the DNA isnot required.

A Waters Action Analyzer was outfitted with a column, as described inExamples 1 and 2, containing 0.5 μm titanium frits. The HPLC instrumentwas a low-pressure mixing quaternary gradient system that containedpolymeric inlet filters and PEEK pump heads, frits, and tubing. Theinlet and outlet check valves were also PEEK, except for a ceramic seatand sapphire ball. A Waters 484 detector was used, with the detectionwavelength set to 254 nm.

In this example, all of the flow paths were either titanium, sapphire,ceramic, or PEEK, except for the column body, which was 316 stainlesssteel. The 316 stainless steel body was passivated with dilute nitricacid prior to packing of the chromatographic material. The 316 stainlesssteel column body was preferred for use in this example because titaniumcolumn bodies were not available with smooth inside walls, and PEEKcolumn bodies flexed during the packing process, leading to lessefficient column beds.

EXAMPLE 9

The effect of acid wash treatment of the PEEK frits used in packing thecolumn on the performance of the HPLC system was demonstrated in thisexample. Using the Waters Action Analyzer and a packed column havinguntreated PEEK frits (Part no. 7100-052-2, Isolation Technologies,Inc.), FIG. 9 (first injection) and FIG. 10 (fifth injection on the samecolumn) show the high resolution separation of DNA restriction fragmentsusing octadecyl modified, nonporouspoly(ethylvinylbenzene-divinylbenzene) beads. The experiment wasconducted under the following conditions: Column: 50×4.6 mm i.d.; mobilephase 0.1 M TEAA, pH 7.2; gradient: 35-55% acetonitrile in 3 min, 55-65%acetonitrile in 7 min, 65% acetonitrile for 2.5 min; 100% acetonitrilefor 1.5 min. back to 35% in 1 min. The flow rate was 0.75 mL/min;detection UV at 260 nm; column temp. 53° C.; p=2200 psi. The sample was5 μl (=0.20 μg pUC18 DNA-Hae III digest).

The PEEK frits from the column used in FIG. 10 were removed andsubjected to an acid wash treatment consisting of sonication in 30%nitric acid for about 60 min, followed by sonication in distilled wateruntil pH=7. The separation procedure was repeated using these acidwashed frits and, as shown in FIG. 11, this led to a major improvementin separation performance although the intensity of the 80 bp and the102 bp fragments was still not satisfactory. As seen in other examples,the degradation of the separation was apparent for the smaller fragmentsrather than the larger fragments. In addition, this degradation appearedto be sequence dependent. Fragments with base pair size 267 and 458 weresmaller than expected relative to fragments eluting immediately prior toand after these fragments. These data suggest that although metal ioncontamination is a factor, there may be more than one mechanism formetals to interfere in MIPC.

For the chromatogram shown in FIG. 12, the separation procedure wasrepeated using PEEK frits which were washed using the following process:The frits from the column used to generate the chromatogram of FIG. 11were subjected to an acid wash treatment consisting of sonication in 30%nitric acid for about 60 min, followed by sonication in distilled wateruntil pH=7. These frits were then sonicated with HCl (36%) for about 5minutes. The chromatogram in FIG. 12 was similar to that seen in FIG.11.

The use of titanium frits (Isolation Technologies, Hopedale, Mass.) isshown in FIG. 13 where the separation conditions were the same as forFIG. 9.

In FIG. 14, the separation as carried out in FIG. 9 was repeated butusing a new column packed using untreated PEEK frits from another source(Part no. A702, Upchurch Scientific, Oak Harbor, Wash.). Thechromatogram in FIG. 14 shows the performance of these frits. Althoughthe overall performance and especially the selectivity is almostidentical to the separation shown in FIG. 13, one major difference canbe detected in that the intensity of fragment 458 bp (peak #8) isclearly decreased.

EXAMPLE 10

The effect of acid wash treatment of the PEEK frits used in packing thecolumn on the separation of single stranded DNA was demonstrated in thisexample. Using the Waters Action Analyzer and a packed column havingtitanium frits, FIG. 15 shows the high resolution separation of DNArestriction fragments using octadecyl modified, nonporouspoly(ethylvinylbenzene-divinylbenzene) beads. The experiment wasconducted under the following conditions: Column: 50×4.6 mm i.d.; mobilephase 0.1 M TEAA, pH 7.2; gradient: 20-80% acetonitrile in 16 min,80-100% acetonitrile in 1.5 min, 100-20% acetonitrile in 1 min. The flowrate was 0.75 mL/min; detection UV at 260 nm; column temp. 51° C.;p=2200 psi. The sample was 5 μl (20-mer oligonucleotide).

As shown in FIG. 15, the column was capable of resolving failuresequences very easily. When the same oligonucleotide was separated on acolumn having PEEK frits as shown in FIG. 16, the oligonucleotide stillappeared at 6 minutes, but the separation performance was deteriorated.Almost all of the smaller peaks (failure sequences) have disappeared dueto the influence of the PEEK frit. The performance of theoligonucleotide separation improved slightly, as shown in FIG. 17, whenusing a column having frits which were treated with HNO₃ and HCl.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention can be practiced otherwise than as specifically describedherein.

All references cited herein are hereby incorporated by reference intheir entirety.

What is claimed is:
 1. A method for separating a mixture ofpolynucleotide fragments during passage through a liquid chromatographiccolumn containing a separation bed comprising Matched Ion PolynucleotideChromatography separation particles, wherein the method comprises:supplying and feeding process solutions entering the column withcomponents having process solution-contacting surfaces which contactprocess solutions held therein or flowing therethrough, wherein saidprocess solution-contacting surfaces are material which does not releasemultivalent cations into aqueous solutions held therein or flowingtherethrough, whereby the column is protected from contamination of thecontents thereof by multivalent cations capable of interfering withpolynucleotide separation, and wherein the polynucleotide fragments areseparated by Matched Ion Polynucleotide Chromatography.
 2. The method ofclaim 1 wherein said multivalent cations are capable of binding DNA. 3.The method of claim 2 wherein said multivalent cations are selected fromthe group consisting of Fe(III), Cr(III), colloidal metal contaminants,and combinations thereof.
 4. The method of claim 3 wherein saidmultivalent cations comprise Fe(III).
 5. The method of claim 1 whereinsaid multivalent cations are selected from the group consisting ofFe(III), Cr(III), colloidal metal contaminants, and combinationsthereof.
 6. The method of claim 5 wherein said multivalent cationscomprise Fe(III).
 7. The method of claim 5 wherein said multivalentcations comprise Cr(III).
 8. The method of claim 1 wherein saidpolynucleotide fragments are separated based on the size of saidpolynucleotide fragments.
 9. The method of claim 1 wherein saidpolynucleotide fragments are separated based on the polarity of saidpolyucleotide fragments.
 10. The method of claim 1, wherein saidsurfaces are comprised of material selected from the group consisting oftitanium, coated stainless steel, sapphire, ceramic, and organicpolymer.
 11. The method of claim 10 wherein said surfaces comprise PEEK.12. The method of claim 1, wherein said surfaces have been subjected toa multivalent cation removal treatment.
 13. The method of claim 12wherein said treatment comprises contacting said surfaces with anaqueous solution containing a member selected from the group consistingof nitric acid, phosphoric acid, pyrophosphoric acid, chelating agent,and combinations thereof.
 14. The method of claim 13 wherein saidcomponents comprise frits.
 15. The method of claim 13 wherein saidchelating agent comprises EDTA.
 16. The method of claim 1 wherein saidprocess solutions comprise mobile phase.
 17. The method of claim 1,wherein said particles have been subjected to an acid wash treatment.18. The method of claim 1 wherein said process solutions include mobilephase additive present in sufficient amount to capture said multivalentcation contaminants.
 19. The method of claim 18 wherein said multivalentcation contaminants are selected from the group consisting of Fe(III),Cr(III), colloidal metal contaminants, and combinations thereof.
 20. Themethod of claim 18 wherein said multivalent cation contaminants compriseFe(III).
 21. The method of claim 20 wherein said additive compriseschelating agent.
 22. The method of claim 21 wherein said agent comprisesEDTA.
 23. The method of claim 22 wherein said polynucleotide fragmentscomprise RNA.
 24. The method of claim 22 wherein said polynucleotidefragments comprise single-stranded DNA.
 25. The method of claim 1including degassing means for removing oxygen from said processsolutions.
 26. The method of claim 1 wherein said polynucleotidefragments comprise RNA.
 27. The method of claim 1 wherein saidpolynucleotide fragments comprise single-stranded DNA.
 28. The method ofclaim 1, wherein the polynucleotide fragments comprise double-strandedfragments.
 29. The method of claim 1, wherein the polynucleotidefragments comprise single-stranded fragments.
 30. The method of claim 1,wherein the polynucleotide fragments comprise 5 or more base pairs. 31.The method of claim 1, wherein said separation particles comprisealkylated non-porous polymer beads having an average diameter of about1-100 microns.
 32. The method of claim 1, wherein said particlescomprise silica and wherein said process solutions comprise EDTA.
 33. Amethod for separating a mixture of polynucleotide fragments duringpassage through a liquid chromatographic column containing a separationbed comprising Matched Ion Polynucleotide Chromatography particles,wherein the method comprises: supplying and feeding process solutionsentering the column with components having process solution-contactingsurfaces which contact process solutions held therein or flowingtherethrough, wherein said process solution-contacting surfaces arematerial which does not release multivalent cations into aqueoussolutions held therein or flowing therethrough, whereby the column isprotected from contamination of the contents thereof by multivalentcations capable of interfering with polynucleotide separation, whereinthe polynucleotide fragments are separated by Matched Ion PolynucleotideChromatography, and wherein said multivalent cations comprise Fe(III).34. The method of claim 33, wherein said surfaces have been subjected toa multivalent cation removal treatment.
 35. The method of claim 34wherein the treatment comprises contacting said surfaces with an aqueoussolution containing a member selected from the group consisting ofnitric acid, phosphoric acid, pyrophosphoric acid, and chelating agents.36. The method of claim 33 wherein said process solutions include amobile phase additive present in sufficient amount to capturemultivalent cation contaminants capable of interfering withpolynucleotide separation.
 37. The method of claim 36 wherein saidadditive comprises a chelating agent.
 38. The method of claim 37 whereinsaid separation particles comprise silica.
 39. The method of claim 38wherein said agent comprises EDTA.
 40. The method of claim 33, whereinthe polynucleotide fragments comprise double-stranded fragments.
 41. Themethod of claim 33, wherein the polynucleotide fragments comprise 5 ormore base pairs.
 42. The method of claim 33 wherein said polynucleotidefragments comprise RNA.
 43. The method of claim 33 wherein saidpolynucleotide fragments comprise single-stranded DNA.
 44. The method ofclaim 33 wherein said surfaces are comprised of material selected fromthe group consisting of titanium, coated stainless steel, sapphire,ceramic, and organic polymer.
 45. A method for separating a mixture ofpolynucleotide fragments during passage through a liquid chromatographiccolumn containing a separation bed comprising Matched Ion PolynucleotideChromatography separation particles, wherein the method comprises:supplying and feeding process solutions entering the column withcomponents having process solution-contacting surfaces which contactprocess solutions held therein or flowing therethrough, wherein saidprocess solution-contacting surfaces are material which does not releasemultivalent cations into aqueous solutions held therein or flowingtherethrough, whereby the column is protected from multivalent cationcontamination of the contents thereof, wherein the polynucleotidefragments are separated by Matched Ion Polynucleotide Chromatography,and wherein said multivalent cations are selected from the groupconsisting of Fe(III), Cr(III), colloidal metal contaminants, andcombinations thereof.
 46. The method of claim 45 wherein said processsolutions include a mobile phase additive present in sufficient amountto capture essentially any of said multivalent cations, and wherein saidadditive comprises a chelating agent.
 47. The method of claim 46 whereinsaid chelating agent comprises EDTA.
 48. The method of claim 47 whereinsaid separation particles comprise silica.
 49. The method of claim 46wherein said multivalent cations comprise contaminants capable ofinterfering with polynucleotide separation.
 50. A method for separatinga mixture of RNA fragments during passage through a liquidchromatographic column containing a separation bed comprising reversephase separation particles, wherein the method comprises: supplying andfeeding process solutions entering the column with components havingprocess solution-contacting surfaces which contact process solutionsheld therein or flowing therethrough, wherein said processsolution-contacting surfaces are material which does not releasemultivalent cations into aqueous solutions held therein or flowingtherethrough, whereby the column is protected from contamination of thecontents thereof by multivalent cations selected from the groupconsisting of Fe(III), Cr(III), colloidal metal contaminants andcombinations thereof, wherein said particles comprise silica, whereinsaid particles are substantially free from multivalent cations selectedfrom the group consisting of Fe(III), Cr(III), colloidal metalcontaminants, and combinations thereof, wherein the RNA fragments areseparated by reverse phase ion pair chromatography, and wherein saidprocess solutions comprise EDTA.
 51. The method of claim 50 wherein saidcomponents comprise titanium or PEEK frits.
 52. A method for separatinga mixture of single-stranded DNA fragments during passage through aliquid chromatographic column containing a separation bed comprisingreverse phase separation particles, wherein the method comprises:supplying and feeding process solutions entering the column withcomponents having process solution-contacting surfaces which contactprocess solutions held therein or flowing therethrough, wherein saidprocess solution-contacting surfaces are material which does not releasemultivalent cations into aqueous solutions held therein or flowingtherethrough, whereby the column is protected from contamination of thecontents thereof by multivalent cations selected from the groupconsisting of Fe(III), Cr(III), colloidal metal contaminants, andcombinations thereof, wherein said particles comprise silica, whereinsaid particles are substantially free from multivalent cations selectedfrom the group consisting of Fe(III), Cr(III), colloidal metalcontaminants, and combinations thereof, wherein the single-stranded DNAfragments are separated by reverse phase ion pair chromatography, andwherein said process solutions comprise EDTA.
 53. The method of claim 52wherein said components comprise titanium or PEEK frits.
 54. A methodfor separating a mixture of double-stranded DNA fragments during passagethrough a liquid chromatographic column containing a separation bedcomprising reverse phase separation particles, wherein the methodcomprises: supplying and feeding process solutions entering the columnwith components having process solution-contacting surfaces whichcontact process solutions held therein or flowing therethrough, whereinsaid process solution-contacting surfaces are material which does notrelease multivalent cations into aqueous solutions held therein orflowing therethrough, whereby the column is protected from contaminationof the contents thereof by multivalent cations selected from the groupconsisting of Fe(III), Cr(III), colloidal metal contaminants, andcombinations thereof, wherein said particles comprise silica, whereinsaid particles are substantially free from multivalent cations selectedfrom the group consisting of Fe(III), Cr(III), colloidal metalcontaminants, and combinations thereof, wherein the double-stranded DNAfragments are separated by reverse phase ion pair chromatography, andwherein said process solutions comprise EDTA.
 55. The method of claim 54wherein said components comprise titanium or PEEK frits.